Peptides for the treatment of neurodegenerative diseases

ABSTRACT

An isolated peptide comprising a Huntingtin (Htt) amino acid sequence being no longer than 15 amino acids, wherein said Htt amino acid sequence comprises the sequence X 1 X 2 X 3 X 4  X 5 , wherein X 1  is a hydrophobic amino acid or threonine, X 2  is a hydrophobic amino acid, X 3  is a hydrophobic amino acid, X 4  is an acidic amino acid and X 5  is selected from the group consisting of glycine, serine and alanine, the peptide capable of specifically inhibiting the activity of caspase 6.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/433,047 filed on Apr. 2, 2015, which is a National Phase of PCTPatent Application No. PCT/IL2013/050196 having International FilingDate of Mar. 5, 2013, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 61/711,309 filed onOct. 9, 2012. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 68935 SequenceListing.txt, created on Jan. 12,2017, comprising 16,384 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to peptides for the treatment ofneurodegenerative disorders and more specifically for the treatment ofHuntington's disease (HD).

Huntington's disease (HD) is an autosomal dominant inheritedneurodegenerative disease. The average age of onset is at 30-50 years ofage. It is characterized by the progressive deterioration of cognitiveand motor functions, with a fatal outcome after approximately 10-15years of onset. HD prevalence varies between 0.5 per 100,000 in Japan,to 5-15 per 100,000 in USA and Europe. The disease was first describedin 1872 by Dr. George Huntington, who published the first description ofthree characteristics of the disease: The heredity, the “tendency toinsanity”, as he observed, and age related onset.

The mutant protein involved in HD, was discovered in 1993 and was namedHuntingtin (Htt). Htt is a large (340-350 kD, 3144 aa) protein that wasshown to be involved in a number of cellular functions such astranscription, gastrulation, neurogenesis, neurotransmission, axonaltransport, neural positioning, and apoptosis. In healthy individuals,Htt contains between 6 to 34 polyQ repeats in the N-terminus (FIG. 6).In the mutant form of Htt (mHtt), there is an extension of the polyQrepeats; a patient carrying over 40 glutamine repeats will certainlydevelop HD. The polyQ expansion results in the selective loss ofGABAergic medium spiny striatal (MSN) neurons as well as glutamatergiccortical neurons that project into the striatum. The loss of theGABAergic MSN results in a lack of inhibitory signals from the striatumto the globus pallidus and the substantia nigra, and therefore inducesexcitatory signals to the neocortex. This disequilibrium is consideredto be the cause for the involuntary movement symptom of the disease.

MHtt expression leads to the formation of PolyQ aggregates andinclusions, transcriptional dysregulation, excessive stimulation ofglutamate receptors leading to excitotoxic neuronal damage, and theinduction of apoptosis. The reduction of normal Htt activity may alsocontribute to disease manifestation, since normal Htt plays an importantrole in neuronal protection and survival and neurogenesis.

Htt was the first neuronal protein discovered to be the subject of aproteolytic cleavage by caspases. Caspase cleavage of Htt occurs atdefined sites for caspase-3 at amino acids 513 and 552, caspase-2 atamino acid 552, and caspase-6 at amino acid 586 (FIG. 6). Additionally,there are two caspase-3 consensus sites at amino acids 530 and 589 thatappear to be silent. Some other proteases, such as calpains, participatein Htt cleavage (FIG. 6).

Although the cleavage occur both in normal and mutant Htt, the mutantform is more susceptible to proteolysis and generates N-terminusfragments that aggregate in the cytoplasm and nucleus of neuronal cells,preceding neurodegeneration. These toxic fragments cause additionalactivation of caspase-6, thus creating a positive feedback cycle ofcaspase activation, and induction of apoptosis.

The significance of mHtt proteolysis in HD pathogenesis is manifested bythe fact that inhibiting caspase cleavage of Htt reduces toxicity andaggregates formation.

In order to reduce mHtt proteolysis, synthetic caspase inhibitors weretested on different experimental models, and indeed showed the reductionof mHtt toxicity induced in neuronal cultures and in HD animal models[Kim, M. et al., The Journal of Neuroscience, 1999, 19, 964-973: Ona, V.O et al. Nature, 1999, 399, 263-267]. These synthetic inhibitors arepseudo substrates for active caspases. They are based on small, usually3-4 aa long peptides, conjugated to carboxyterminal groups such aschloromethyl ketone (cmk), fluoromethyl ketone (fmk), or aldehyde (cho).These groups enable them to act as competitive inhibitors, increase theaffinity to caspases and improve cell permeability and stability. Thesecell permeable inhibitors alkylate the active site cysteine of caspasesand irreversibly block apoptosis by preventing caspase activation,substrate cleavage, and DNA ladder formation. However, most syntheticcaspase inhibitors are hydrophobic and not very permeable, and couldcause nonspecific toxic effects when added at concentrations required toinhibit intracellular caspases [Ona, V. O et al. Nature, 1999, 399,263-267: Frydrych, I.; Toxicology in Vitro, Proceedings of theScandinavian Society of Cell Toxicology 2007 Workshop, 2008, 22,1356-1360; Zhu, S.; et al., Cell Death and Dis, Macmillan PublishersLimited, 2011, 2, e115; Chauvier, D. Cell Death Differ, 2006, 14,387-391]. Experiments in knockout mice indicated that caspase-6deficiency is not fatal or causes severe toxic effects.

Garcio et al (The Journal of Biological Chemistry, 273, (4): 371-376)teaches inhibition of human caspases by peptide based and macromolecularinhibitors. Nyormoi O et al., (Apoptosis. 2003 August; 8(4):371-6)teaches a synthetic peptide inhibitor of caspase 6.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated peptide comprising a Huntingtin (Htt)amino acid sequence being no longer than 15 amino acids, wherein the Httamino acid sequence comprises the sequence X₁X₂X₃X_(4,)X₅ (SEQ ID NO:33) wherein X₁ is a hydrophobic amino acid or threonine, X₂ is ahydrophobic amino acid, X₃ is a hydrophobic amino acid, X₄ is an acidicamino acid and X₅ is selected from the group consisting of glycine,serine and alanine, the peptide capable of specifically inhibiting theactivity of caspase 6

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a caspase 6 mediated disease in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the isolated peptidedescribed herein, thereby treating the caspase 6 mediated disease.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedpeptide described herein and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the X₁ is selected fromthe group consisting of isoleucine, valine, threonine, leucine, alanine,methionine, phenylalanine, tyrosine and tryptophan.

According to some embodiments of the invention, the X₄ is aspartic acidor glutamic acid.

According to some embodiments of the invention, the X₂ or X₃ is selectedfrom the group consisting of isoleucine, valine, leucine, alanine,methionine, phenylalanine, tyrosine and tryptophan.

According to some embodiments of the invention, the X₁ is isoleucine.

According to some embodiments of the invention, the X₂ is valine.

According to some embodiments of the invention, the X₃ is leucine.

According to some embodiments of the invention, the X₄ is aspartic acid.

According to some embodiments of the invention, the Htt amino acidsequence comprises the sequence as set forth in SEQ ID NO: 5.

According to some embodiments of the invention, the Htt amino acidsequence comprises the sequence X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 34), wherein X₆is selected from the group consisting of threonine, serine, asparaginesand glutamine.

According to some embodiments of the invention, the Htt amino acidsequence comprises the sequence X₁X₂X₃X₄X₅X₆X₇, (SEQ ID NO: 35) whereinX₇ is aspartic acid or glutamic acid.

According to some embodiments of the invention, the Htt amino acidsequence comprises the sequence X₁X₂X₃X₄X₅X₆X₇X₈, (SEQ ID NO: 36)wherein X₈ is selected from the group consisting of serine, asparagines,threonine and glutamine.

According to some embodiments of the invention, the Htt amino acidsequence comprises the amino acid sequence as set forth in SEQ ID NOs:2-5.

According to some embodiments of the invention, the Htt amino acidsequence is attached to a cell penetrating agent.

According to some embodiments of the invention, the cell penetratingagent is a cell penetrating peptide agent.

According to some embodiments of the invention, the cell penetratingpeptide agent comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7-15 and 16.

According to some embodiments of the invention, the cell penetratingpeptide agent is attached directly to the Htt amino acid sequence.

According to some embodiments of the invention, the cell penetratingpeptide agent is attached to the Htt amino acid sequence via a linkingamino acid sequence.

According to some embodiments of the invention, the linking amino acidsequence comprises a disulfide bridge.

According to some embodiments of the invention, the linking amino acidsequence is attached to the N terminal of the Htt amino acid sequence.

According to some embodiments of the invention, the linking amino acidsequence is attached to the C terminal of the cell penetrating peptideagent.

According to some embodiments of the invention, the linking amino acidsequence comprises the sequence SSE.

According to some embodiments of the invention, the isolated peptidecomprises the sequence as set forth in SEQ ID NOs: 6 or 28.

According to some embodiments of the invention, the isolated peptide isno longer than 25 amino acids.

According to some embodiments of the invention, the peptide inhibits theactivity of caspase 6 to a greater extent than it inhibits the activityof caspase 3.

According to some embodiments of the invention, the caspase 6 mediateddisease is a neurodegenerative disease.

According to some embodiments of the invention, the neurodegenerativedisease is selected from the group consisting of Huntington's Disease(HD), Alzheimer's Disease (AD), aging, and stroke.

According to some embodiments of the invention, the neurodegenerativedisease is HD.

According to some embodiments of the invention, the isolated peptide isfor treating a caspase 6 mediated disease.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1F—ED11 inhibits caspase-6 but not caspase-3 activity in apurified caspase activity assay: A-B. Reaction development over time isinhibited in the presence of ED11 compared to vehicle or TAT-onlycontrol (A) in a dose-dependent matter (B). C. Michaelis-Menten curveshows classic competitive inhibition as Vmax stays approximately thesame and Km shows a 3.3 fold increase. D. ED11 does not inhibitcaspase-3 in a fluorescent caspsae-3 inhibition assay. E-F ED11 inhibitscaspase-6 but not caspase-3, unlike the synthetic caspase inhibitorZ-VAD-FMK which inhibits both caspase-6 and caspase-3. Error barsrepresent S.E.M.

FIGS. 2A-2C—ED11 does not influence cell viability, proliferation orcell cycle status in a basal state: A. Cell viability was measured usingALAMAR blue assay after 48 hours incubation with ED11, demonstrating noreduction in cell viability. B. Cell proliferative state was measuredusing BrdU proliferation assay, indicating no proliferative effect ofED11. C. Cell cycle is not altered as a result of ED11 O.N. incubation.

FIGS. 3A-3D—ED11 inhibits in-cell caspase-6 activity following glutamateexposure in SH-SY5Y cells, and protects SH-SY5Y cells viability fromglutamate induced toxicity: A-B. Annexin V PI apoptosis assay shows asignificant reduction of the apoptotic process caused by glutamate incells treated with ED11. Graph represents the means of 4 independentexperiments. C. Cell viability evaluation as measured by Alamar blueviability assay shows ED11 protects cell viability from glutamateinsult. D. Glutamate increases caspase-6 activity as measured by in-cellFLICA caspase-6 activity assay. This increase in caspase-6 activity ishighly moderated when cells are pre-treated with ED11. Error barsrepresent S.E.M. (B) One-way ANOVA, (D) Two-way ANOVA. *P<0.05**P<0.001.

FIGS. 4A-4D—ED11 protects PC12 cells from mutant Huntingtin's toxicity:PC12 cells were induced to express mutant Huntingtin (145Q) by PA andwere grown under serum deprivation condition. A. Cell viability wasmeasured by Alamar blue viability assay, 48 and 72 hours post induction,demonstrating that ED11 protects cell viability from mHtt toxicity. B.Cell death measured by LDH release, 48 and 72 hours after mHttinduction, was highly attenuated by ED11. C-D. Representative microscopypictures (C) and area scan quantification (D) show ED11 inhibits in-cellcaspase-6 activity initiated by mHtt. Error bars represents S.E.M. (A-B)Two-way ANOVA, (D) One-way ANOVA. *P<0.05 **P<0.01.

FIGS. 5A-5B—ED11 prevents mHtt direct cleavage by caspase-6: BACHDstriata protein extracts were incubated with caspase-6 for 20 minutes in37° C. and proteins were assayed using standard western blot assay. Lanedescription: 1-2: Wild type control extracts. 3-4: BACHD without C6control. 5-6: BACHD with C6+DMSO 7-8: BACHD with C6+ED11. A-B.Incubation with Anti-Htt 4-19 antibody (A) or MAB 2166 (B) showedcaspase-6 fragmentation of mHtt (*) is blocked in the presence of ED11.

FIG. 6—Huntingtin structure and cleavage sites diagram (modified fromCattaneo et al. Nat Rev Neurosci, 2005, 6, 919-930. (Q)n indicates thepolyglutamine tract, which is followed by a polyproline sequence, (P)n.The arrows indicate the caspase cleavage sites and their amino acidpositions, and the blue arrowheads the calpain cleavage sites and theiramino acid position. Green and orange arrowheads point to theapproximate amino acid regions for protease cleavage. NES is the nuclearexport signal. Blue lines zoom in the amino acid sequence most relevantfor caspase cleavage of HTT (Adapted from Warby et-al., Human MolecularGenetics, 2008, 17, 2390-2404). The illustrated amino acid sequence isset forth in SEQ ID NO: 32.

FIGS. 7A-7C are bar graphs illustrating that the ED11 peptide protectsHuntington's disease mouse model BACHD from motor deterioration astested in the accelerating Rotarod test.

FIG. 8 is a bar graph illustrating that the amino acids at positions X1and X4 of the ED11 peptide are important for the caspase 6 inhibitoryactivity.

(SEQ ID NO: 28) ED11 S-TAT: YGRKKRRSSEIVLDGTDN; (SEQ ID NO: 29)A1: YGRKKRRSSEAVLDGTDN; (SEQ ID NO: 30) A2: YGRKKRRSSEIVLAGTDN;(SEQ ID NO: 31) A3: YGRKKRRSSEIVLDGTAN.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to peptides for the treatment ofneurodegenerative disorders and more specifically for the treatment ofHuntington's disease (HD).

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Huntington's disease (HD) is an autosomal dominant inheritedneurodegenerative disease. It is characterized by the progressivedeterioration of cognitive and motor functions, with a fatal outcomeafter approximately 10-15 years of onset. When the mutated proteinhuntingtin carries over 40 glutamine repeats at the N-terminal, HD willoccur. The precise pathogenic mechanism of mutant Huntingtin is unknown.However, it was shown that caspase-6 plays a key role in the apoptoticevents seen in HD. Caspase-6 is activated early in the disease progress,inducing different cellular dysfunctions. Importantly, caspase-6proteolysis of mutant huntingtin is known to be an essential process ingenerating toxic N-terminal fragments, which leads to neurodegenerationand appearance of disease symptoms. Therefore, inhibiting caspase-6activity was suggested as a promising therapeutic target for reducingmutant huntingtin toxicity, and rescuing neuronal cells fromdegeneration.

The present inventors constructed huntingtin cleavage site basedpeptides fused with a cell penetrating peptide (CPP) to be used as atherapeutic for treating Huntingtin.

The present inventors discovered that a minimum core sequence of 5 aminoacids of the caspase cleavage site was required for the peptide to havetherapeutic activity. Several candidate peptides comprising this coresequenced were synthesized and tested biochemically. One of thepeptides, (ED11), was composed of 11 amino acids (aa) derived fromhuntingtin sequence fused with 13 aa of the CPP (SEQ ID NO: 6). Thispeptide significantly and specifically reduced caspase-6 activity asindicated by caspase-6 activity assay in a cell free system (FIGS.1A-1F). Moreover, in neuronal cells this peptide inhibited caspase-6activity, and protected the cells from glutamate toxicity, an importantfactor in the pathogenesis of HD (FIGS. 3A-3D). Furthermore, in PC12cells, expressing mutant huntingtin, the peptide markedly reduced theserum deprivation induced toxicity, compared to wild-type PC12 cells(FIGS. 4A-4D). Whilst further reducing the present invention topractice, the present inventors demonstrated that when BACHD mice brainstriatum extracts were incubated with caspase-6, the addition of thepeptide significantly inhibited human mutant huntingtin fragmentation.

In vivo administration of the peptide to BACHD mice had therapeuticeffects as assayed by rotarod performance (FIGS. 7A-7C). Importantly,the peptide was found to be non-toxic to neuronal cells even at highdoses (FIGS. 2A-2C).

The present inventors propose the use of the above described peptidesfor the treatment of caspase 6 related diseases in general andHuntingtin Disease in particular.

Thus, according to one aspect of the present invention there is providedan isolated peptide comprising a Huntingtin (Htt) amino acid sequencebeing no longer than 15 amino acids, wherein the Htt amino acid sequencecomprises the sequence X₁X₂X₃X₄X₅ (SEQ ID NO: 34) wherein X₁ is ahydrophobic amino acid or threonine, X₂ is a hydrophobic amino acid, X₃is a hydrophobic amino acid, X₄ is an acidic amino acid and X₅ isselected from the group consisting of glycine, serine and alanine, thepeptide capable of specifically inhibiting the activity of caspase 6.

The term “peptide” as used herein refers to a polymer of natural orsynthetic amino acids, encompassing native peptides (either degradationproducts, synthetically synthesized polypeptides or recombinantpolypeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are polypeptideanalogs, which may have, for example, modifications rendering thepeptides even more stable while in a body or more capable of penetratinginto cells.

Such modifications include, but are not limited to N terminusmodification, C terminus modification, polypeptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Polypeptide bonds (—CO—NH—) within the polypeptide may be substituted,for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N-—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the polypeptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the polypeptides of the present invention mayalso include one or more modified amino acids or one or more non-aminoacid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids (stereoisomers).

Tables 1 and 2 below list naturally occurring amino acids (Table 1) andnon-conventional or modified amino acids (Table 2) which can be usedwith the present invention.

TABLE 1 Three-Letter Amino Acid Abbreviation One-letter Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 2 Non-conventional amino Non-conventional amino acid Code acidCode ornithine Orn hydroxyproline Hyp α-aminobutyric acid Abuaminonorbornyl- Norb carboxylate D-alanine Dala aminocyclopropane- Cprocarboxylate D-arginine Darg N-(3- Narg guanidinopropyl)glycineD-aparagine Dasn N-(carbamylmethyl)glycine Nasn D-aspartic acid DaspN-(carboxymethyl)glycine Nasp D-cysteine Dcys N-(thiomethyl)glycine NcysD-glutamine Dgln N-(2-carbamylethyl)glycine Ngln D-glutamic acid DgluN-(2-carboxyethyl)glycine Nglu D-histidine DhisN-(imidazolylethyl)glycine Nhis D-isoleucine DileN-(1-methylpropyl)glycine Nile D-leucine Dleu N-(2-methylpropyl)glycineNleu D-lysine Dlys N-(4-aminobutyl)glycine Nlys D-methionine DmetN-(2-methylthioethyl)glycine Nmet D-ornithine DornN-(3-aminopropyl)glycine Norn D-phenylalanine Dphe N-benzylglycine NpheD-proline Dpro N-(hydroxymethyl)glycine Nser D-serine DserN-(1-hydroxyethyl)glycine Nthr D-threonine Dthr N-(3-indolylethyl)glycine Nhtrp D-tryptophan Dtrp N-(p-hydroxyphenyl)glycine NtyrD-tyrosine Dtyr N-(1-methylethyl)glycine Nval D-valine DvalN-methylglycine Nmgly D-N-methylalanine Dnmala L-N-methylalanine NmalaD-N-methylarginine Dnmarg L-N-methylarginine Nmarg D-N-methylasparagineDnmasn L-N-methylasparagine Nmasn D-N-methylasparatate DnmaspL-N-methylaspartic acid Nmasp D-N-methylcysteine DnmcysL-N-methylcysteine Nmcys D-N-methylglutamine Dnmgln L-N-methylglutamineNmgln D-N-methylglutamate Dnmglu L-N-methylglutamic acid NmgluD-N-methylhistidine Dnmhis L-N-methylhistidine NmhisD-N-methylisoleucine Dnmile L-N-methylisolleucine NmileD-N-methylleucine Dnmleu L-N-methylleucine Nmleu D-N-methyllysine DnmlysL-N-methyllysine Nmlys D-N-methylmethionine Dnmmet L-N-methylmethionineNmmet D-N-methylornithine Dnmorn L-N-methylornithine NmornD-N-methylphenylalanine Dnmphe L-N-methylphenylalanine NmpheD-N-methylproline Dnmpro L-N-methylproline Nmpro D-N-methylserine DnmserL-N-methylserine Nmser D-N-methylthreonine Dnmthr L-N-methylthreonineNmthr D-N-methyltryptophan Dnmtrp L-N-methyltryptophan NmtrpD-N-methyltyrosine Dnmtyr L-N-methyltyrosine Nmtyr D-N-methylvalineDnmval L-N-methylvaline Nmval L-norleucine Nle L-N-methylnorleucineNmnle L-norvaline Nva L-N-methylnorvaline Nmnva L-ethylglycine EtgL-N-methyl-ethylglycine Nmetg L-t-butylglycine TbugL-N-methyl-t-butylglycine Nmtbug L-homophenylalanine Hphe L-N-methyl-Nmhphe homophenylalanine α-naphthylalanine AnapN-methyl-α-naphthylalanine Nmanap penicillamine PenN-methylpenicillamine Nmpen γ-aminobutyric acid GabuN-methyl-γ-aminobutyrate Nmgabu cyclohexylalanine ChexaN-methyl-cyclohexylalanine Nmchexa cyclopentylalanine CpenN-methyl-cyclopentylalanine Nmcpen α-amino-α-methylbutyrate AabuN-methyl-α-amino-α- Nmaabu methylbutyrate α-aminoisobutyric acid AibN-methyl-α- Nmaib aminoisobutyrate D-α-methylarginine DmargL-α-methylarginine Marg D-α-methylasparagine Dmasn L-α-methylasparagineMasn D-α-methylaspartate Dmasp L-α-methylaspartate MaspD-α-methylcysteine Dmcys L-α-methylcysteine Mcys D-α-methylglutamineDmgln L-α-methylglutamine Mgln D-α-methyl glutamic acid DmgluL-α-methylglutamate Mglu D-α-methylhistidine Dmhis L-α-methylhistidineMhis D-α-methylisoleucine Dmile L-α-methylisoleucine MileD-α-methylleucine Dmleu L-α-methylleucine Mleu D-α-methyllysine DmlysL-α-methyllysine Mlys D-α-methylmethionine Dmmet L-α-methylmethionineMmet D-α-methylornithine Dmorn L-α-methylornithine MornD-α-methylphenylalanine Dmphe L-α-methylphenylalanine MpheD-α-methylproline Dmpro L-α-methylproline Mpro D-α-methylserine DmserL-α-methylserine Mser D-α-methylthreonine Dmthr L-α-methylthreonine MthrD-α-methyltryptophan Dmtrp L-α-methyltryptophan Mtrp D-α-methyltyrosineDmtyr L-α-methyltyrosine Mtyr D-α-methylvaline Dmval L-α-methylvalineMval N-cyclobutylglycine Ncbut L-α-methylnorvaline MnvaN-cycloheptylglycine Nchep L-α-methylethylglycine MetgN-cyclohexylglycine Nchex L-α-methyl-t-butylglycine MtbugN-cyclodecylglycine Ncdec L-α-methyl- Mhphe homophenylalanineN-cyclododecylglycine Ncdod α-methyl-α-naphthylalanine ManapN-cyclooctylglycine Ncoct α-methylpenicillamine MpenN-cyclopropylglycine Ncpro α-methyl-γ-aminobutyrate MgabuN-cycloundecylglycine Ncund α-methyl-cyclohexylalanine MchexaN-(2-aminoethyl)glycine Naeg α-methyl-cyclopentylalanine Mcpen N-(2,2-Nbhm N-(N-(2,2-diphenylethyl) Nnbhm diphenylethyl)glycinecarbamylmethyl-glycine N-(3,3- Nbhe N-(N-(3,3-diphenylpropyl) Nnbhediphenylpropyl)glycine carbamylmethyl-glycine 1-carboxy-1-(2,2-diphenylNmbc 1,2,3,4- Tic ethylamino)cyclopropane tetrahydroisoquinoline-3-carboxylic acid phosphoserine pSer phosphothreonine pThr phosphotyrosinepTyr O-methyl-tyrosine 2-aminoadipic acid hydroxylysine

The amino acids of the peptides of the present invention may besubstituted either conservatively or non-conservatively.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered asconservative substitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acid is well documented in the literature known to theskilled practitioner.

When affecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Thosenon-conservative substitutions which fall under the scope of the presentinvention are those which still constitute a peptide havinganti-bacterial properties.

As mentioned, the N and C termini of the peptides of the presentinvention may be protected by function groups. Suitable functionalgroups are described in Green and Wuts, “Protecting Groups in OrganicSynthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachingsof which are incorporated herein by reference. Preferred protectinggroups are those that facilitate transport of the compound attachedthereto into a cell, for example, by reducing the hydrophilicity andincreasing the lipophilicity of the compounds.

These moieties can be cleaved in vivo, either by hydrolysis orenzymatically, inside the cell. Hydroxyl protecting groups includeesters, carbonates and carbamate protecting groups. Amine protectinggroups include alkoxy and aryloxy carbonyl groups, as described abovefor N-terminal protecting groups. Carboxylic acid protecting groupsinclude aliphatic, benzylic and aryl esters, as described above forC-terminal protecting groups. In one embodiment, the carboxylic acidgroup in the side chain of one or more glutamic acid or aspartic acidresidue in a peptide of the present invention is protected, preferablywith a methyl, ethyl, benzyl or substituted benzyl ester.

Examples of N-terminal protecting groups include acyl groups (—CO—R1)and alkoxy carbonyl or aryloxy carbonyl groups (—CO—O—R1), wherein R1 isan aliphatic, substituted aliphatic, benzyl, substituted benzyl,aromatic or a substituted aromatic group. Specific examples of acylgroups include acetyl, (ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—,n-butyl-CO—, sec-butyl-CO—, t-butyl-CO—, hexyl, lauroyl, palmitoyl,myristoyl, stearyl, oleoyl phenyl-CO—, substituted phenyl-CO—,benzyl-CO— and (substituted benzyl)-CO—. Examples of alkoxy carbonyl andaryloxy carbonyl groups include CH3-O—CO—, (ethyl)-O—CO—,n-propyl-O—CO—, iso-propyl-O—CO—, n-butyl-O—CO—, sec-butyl-O—CO—,t-butyl-O—CO—, phenyl-O—CO—, substituted phenyl-O—CO— and benzyl-O—CO—,(substituted benzyl)-O—CO—. Adamantan, naphtalen, myristoleyl, tuluen,biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane,norbornane, Z-caproic. In order to facilitate the N-acylation, one tofour glycine residues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by an amide (i.e., the hydroxyl group at the C-terminus isreplaced with —NH₂, —NHR₂ and —NR₂R₃) or ester (i.e. the hydroxyl groupat the C-terminus is replaced with —OR₂). R₂ and R₃ are independently analiphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or asubstituted aryl group. In addition, taken together with the nitrogenatom, R₂ and R₃ can form a C4 to C8 heterocyclic ring with from about0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examplesof suitable heterocyclic rings include piperidinyl, pyrrolidinyl,morpholino, thiomorpholino or piperazinyl. Examples of C-terminalprotecting groups include —NH₂, —NHCH₃, —N(CH₃)₂, —NH(ethyl),-N(ethyl)₂, —N(methyl) (ethyl), —NH(benzyl), —N(C1-C4 alkyl)(benzyl),—NH(phenyl), —N(C1-C4 alkyl) (phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl),—O-(n-butyl), —O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyland —O-phenyl.

The peptides of the present invention may also comprise non-amino acidmoieties, such as for example, hydrophobic moieties (various linear,branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbonderivatives) attached to the peptides; non-peptide penetrating agents;various protecting groups, especially where the compound is linear,which are attached to the compound's terminals to decrease degradation.Chemical (non-amino acid) groups present in the compound may be includedin order to improve various physiological properties such; decreaseddegradation or clearance; decreased repulsion by various cellular pumps,improve immunogenic activities, improve various modes of administration(such as attachment of various sequences which allow penetration throughvarious barriers, through the gut, etc.); increased specificity,increased affinity, decreased toxicity and the like.

Attaching the amino acid sequence component of the peptides of theinvention to other non-amino acid agents may be by covalent linking, bynon-covalent complexion, for example, by complexion to a hydrophobicpolymer, which can be degraded or cleaved producing a compound capableof sustained release; by entrapping the amino acid part of the peptidein liposomes or micelles to produce the final peptide of the invention.The association may be by the entrapment of the amino acid sequencewithin the other component (liposome, micelle) or the impregnation ofthe amino acid sequence within a polymer to produce the final peptide ofthe invention.

The peptides of the invention may be linear or cyclic (cyclization mayimprove stability). Cyclization may take place by any means known in theart. Where the compound is composed predominantly of amino acids,cyclization may be via N- to C-terminal, N-terminal to side chain andN-terminal to backbone, C-terminal to side chain, C-terminal tobackbone, side chain to backbone and side chain to side chain, as wellas backbone to backbone cyclization. Cyclization of the peptide may alsotake place through non-amino acid organic moieties comprised in thepeptide.

The peptides of the present invention can be biochemically synthesizedsuch as by using standard solid phase techniques. These methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation, classical solution synthesis. Solid phasepolypeptide synthesis procedures are well known in the art and furtherdescribed by John Morrow Stewart and Janis Dillaha Young, Solid PhasePolypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Large scale peptide synthesis is described by Andersson Biopolymers2000; 55(3):227-50.

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

Recombinant techniques may also be used to generate the peptides of thepresent invention. To produce a peptide of the present invention usingrecombinant technology, a polynucleotide encoding the peptide of thepresent invention is ligated into a nucleic acid expression vector,which comprises the polynucleotide sequence under the transcriptionalcontrol of a cis-regulatory sequence (e.g., promoter sequence) suitablefor directing constitutive, tissue specific or inducible transcriptionof the polypeptides of the present invention in the host cells.

In addition to being synthesizable in host cells, the peptides of thepresent invention can also be synthesized using in vitro expressionsystems. These methods are well known in the art and the components ofthe system are commercially available.

Peptides contemplated by the present invention comprise the coresequence IVLD-SEQ ID NO: 1.

Examples of such peptides include:

(SEQ ID NO: 2) IVLDGTDN; (SEQ ID NO: 3) IVLDGTD; (SEQ ID NO: 4) IVLDGT;(SEQ ID NO: 5) IVLDG;

For the peptide having the sequence as set forth in SEQ ID NO: 2, aminoacid “I” may be considered to be in position 1; amino acid “V” may beconsidered to be in position 2; “amino acid “L” may be considered to bein position 3; “amino acid “D” may be considered to be in position 4;amino acid “G” may be considered to be in position 5; amino acid “T” maybe considered to be in position 6; amino acid “D” may be considered tobe in position 7; and amino acid “N” may be considered to be in position8.

Contemplated replacements for any of the amino acids in this sequenceinclude:

-   -   1. The use of Valine (V) as a replacement for Iso-leucine (I) at        position #1;    -   2. The use of Threonine (T) as a replacement for Iso-leucine (I)        at position #1;    -   3. The use of Leucine (L) as a replacement for Iso-leucine (I)        at position #1;    -   4. The use of Glutamic acid (E) as a replacement for Aspartic        acid (D) at position #4;    -   5. The use of Serine (S) as a replacement of Glycine(G) at        position #5;

The use of Alanine(A) as a replacement of Glycine(G) at position #5; or

-   -   6. The use of Glutamic acid (E) as a replacement for Aspartic        acid (D) at position #7.

Additional contemplated replacements for any of the amino acids in thissequence include:

-   -   1. The use of Alanine (A) as a replacement for Iso-leucine (I)        at position #1;    -   2. The use of Methionine (M) as a replacement for        Iso-leucine (I) at position #1;    -   3. The use of Phenylalanine (F) as a replacement for        Iso-leucine (I) at position #1;    -   4. The use of Tyrosine (Y) as a replacement for Iso-leucine (I)        at position #1;    -   5. The use of Tryptopane (W) as a replacement for        Iso-leucine (I) at position #1;    -   6. The use of Leucine (L) as a replacement for Valine (V) at        position #2;    -   7. The use of Iso-Leucine (I) as a replacement for Valine (V) at        position #2;    -   8. The use of Alanine (A) as a replacement for Valine (V) at        position #2;    -   9. The use of Methionine (M) as a replacement for Valine (V) at        position #2;    -   10. The use of Phenylalanine (F) as a replacement for Valine (V)        at position #2;    -   11. The use of Tyrosine (Y) as a replacement for Valine (V) at        position #2;    -   12. The use of Tryptopane (W) as a replacement for Valine (V) at        position #2;    -   13. The use of Valine (V) as a replacement for Leucine (L) at        position #3;    -   14. The use of Iso-Leucine (I) as a replacement for Leucine (L)        at position #3;    -   15. The use of Alanine (A) as a replacement for Leucine (L) at        position #3;    -   16. The use of Methionine (M) as a replacement for Leucine (L)        at position #3;    -   17. The use of Phenylalanine (F) as a replacement for        Leucine (L) at position #3;    -   18. The use of Tyrosine (Y) as a replacement for Leucine (L) at        position #3;    -   19. The use of Tryptopane (W) as a replacement for Leucine (L)        at position #3;    -   20. The use of Serine (S) as a replacement for Threonine (T) at        position #6;    -   21. The use of Aspargine (N) as a replacement for Threonine (T)        at position #6;    -   22. The use of Glutamine (Q) as a replacement for Threonine (T)        at position #6;    -   23. The use of Serine (S) as a replacement for Aspargine (N) at        position #8;    -   24. The use of Threonine (T) as a replacement for Aspargine (N)        at position #8;    -   25. The use of Glutamine (Q) as a replacement for Aspargine (N)        at position #8; or    -   26. D-confirmation of Aspartic acid (D) at position #4 of the        core peptide.

As mentioned, the peptides of this aspect of the present inventioncomprise a caspase 6 inhibitory activity. Methods of analyzing whether apeptide comprises caspase 6 inhibitory activity are known in the art andinclude use of commercial kits such as those available from Promega(catalogue number G0970), Abcom (catalogue number ab39707) and Millipore(catalogue number APT168).

Preferably, the peptides inhibit the activity of caspase 6 to a greaterextent than they inhibit the activity of caspase 3. For example the Kiof the peptide may be at least 2 fold, preferably at least 5 fold lowerfor caspase 6 than for caspase 3. Methods of determining the inhibitoryactivity of the peptides towards caspase 3 are known in the art andinclude the use of kits such as those available from Promega, CellSignal or Sigma.

As mentioned, the Huntingtin (Htt) amino acid sequence is preferably nolonger than 15 amino acids, more preferably no longer than 14 aminoacids, more preferably no longer than 13 amino acids, more preferably nolonger than 12 amino acids, more preferably no longer than 11 aminoacids.

According to another embodiment, the Htt amino acid sequence is nolonger than 10 amino acids, more preferably no longer than 9 aminoacids, more preferably no longer than 8 amino acids.

As mentioned, the peptides described herein may be attached to a cellpenetrating agent.

As used herein the phrase “penetrating agent” refers to an agent whichenhances translocation of any of the attached peptide across a cellmembrane.

According to one embodiment, the penetrating agent is a peptide and isattached to the Huntingtin related peptide (either directly ornon-directly) via a peptide bond.

Typically, peptide penetrating agents have an amino acid compositioncontaining either a high relative abundance of positively charged aminoacids such as lysine or arginine, or have sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids.

Examples of peptide penetrating agents include long and short versionsof TAT (YGRKKRR—SEQ ID NO: 14 and YGRKKRRQRRR—SEQ ID NO: 15) and PTD(RRQRR—SEQ ID NO: 16. By way of non-limiting example, cell penetratingpeptide (CPP) sequences may be used in order to enhance intracellularpenetration. CPPs may include:

SEQ ID NO: 7 GRKKRRQRRRPPQ; SEQ ID NO: 8 GRKKRRQRRRPP; SEQ ID NO: 9GRKKRRQRRRP; SEQ ID NO: 10 GRKKRRQRRR; SEQ ID NO: 11 GRKKRRQRR;SEQ ID NO: 12 GRKKRRQR; SEQ ID NO: 13 GRKKRRQ; SEQ ID NO: 14 YGRKKRR;SEQ ID NO: 15 YGRKKRRQRRR; SEQ ID NO: 16 RRQRR.

According to a particular embodiment, the Htt peptides are attached tothe cell penetrating peptides via a linking moiety.

Examples of linking moieties include but are not limited to a simplecovalent bond, a flexible peptide linker, a disulfide bridge or apolymer such as polyethylene glycol (PEG). Peptide linkers may beentirely artificial (e.g., comprising 2 to 20 amino acid residuesindependently selected from the group consisting of glycine, serine,asparagine, threonine and alanine) or adopted from naturally occurringproteins. Disulfide bridge formation can be achieved, e.g., by additionof cysteine residues, as further described herein below.

Selection of the link between the two peptides should take into accountthat the link should not substantially interfere with the ability of theHtt peptide to inhibit caspase 6 activity or the ability of the cellpenetrating peptide to traverse the cell membrane.

Thus, for example, the linking moiety is optionally a moiety which iscovalently attached to a side chain, an N-terminus or a C-terminus ofthe Htt peptide, as well as to a side chain, an N-terminus or aC-terminus of the cell penetrating peptide.

The linking moiety may be attached to the C-terminus of the Htt peptidemonomer and to the N-terminus of the cell penetrating peptide.

Alternatively, the linking moiety may be attached to the N-terminus ofthe Htt peptide monomer and to the C-terminus of the cell penetratingpeptide.

The linking moiety (or linking peptide) may comprise additional aminoacids from the huntingtin protein.

Thus, according to a particular embodiment, the linking moiety comprisesthe sequence SSE.

The linker may comprise additional amino acids linked together bypeptide bonds which serve as spacers such that the linker does notinterfere with the biological activity of the final compound. The linkeris preferably made up of amino acids linked together by peptide bonds.Thus, in preferred embodiments, the linker is made up of from 1 to 10amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart.

In a more preferred embodiment, besides serine and glutamic acid theamino acids in the linker are selected from glycine, alanine, proline,asparagine and lysine. Even more preferably, besides serine and glutamicacid, the linker is made up of a majority of amino acids that aresterically unhindered, such as glycine and alanine.

According to another embodiment, the linking peptide comprises adisulfide bridge.

Thus, in some embodiments of the invention, each of the peptidescomprises an amino acid sequence as described herein above and furthercomprise at least one cysteine residue, such that the peptides arecovalently linked to one another via a disulfide bridge formed between acysteine residue in one peptide and a cysteine residue in anotherpeptide.

Hereinthroughout, the phrases “disulfide bridge” and “disulfide bond”are used interchangeably, and describe a —S—S— bond.

Examples of peptides which include both the Htt sequence and the cellpenetrating peptide are disclosed below:

SEQ ID NO: 6 GRKKRRQRRRPPQSSEIVLDGTDN; SEQ ID NO: 17GRKKRRQRRRPPQSSEIVLDGTD; SEQ ID NO: 18 GRKKRRQRRRPPQSSEIVLDGT;SEQ ID NO: 19 GRKKRRQRRRPPQSSEIVLDG; SEQ ID NO: 20 GRKKRRQRRRPPQSSEIVLD;SEQ ID NO: 21 GRKKRRQRRRPPQIVLDGTDN; SEQ ID NO: 22GRKKRRQRRRPPSSEIVLDGTDN; SEQ ID NO: 23 GRKKRRQRRRPSSEIVLDGTDN;SEQ ID NO: 24 GRKKRRQRRRSSEIVLDGTDN; SEQ ID NO: 25 GRKKRRQRRSSEIVLDGTDN;SEQ ID NO: 26 GRKKRRQRSSEIVLDGTDN; SEQ ID NO: 27 GRKKRRQSSEIVLDGTDN;

The full length peptide (i.e. Huntington peptide, optional linkingpeptide and optional cell penetrating peptide) is typically no longerthan 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 aminoacids, 29 amino acids or 30 amino acids.

Since the peptides disclosed herein are capable of inhibiting caspase 6,the present inventors propose that they may be used to treat and/orprevent caspase 6 associated diseases, examples of which are providedherein below.

1. chronic degenerative diseases e.g. neurodegenerative diseaseincluding Alzheimer's disease, Huntington's′ disease, Parkinson's′disease, Multiple sclerosis, amyotrophic lateral sclerosis, spinobulbaratrophy, prion disease, dementia

2. epilepsy or epilepstogenes

3. apoptosis during spinal cord injury

4. apoptosis resulting from traumatic brain injury, or to provideneuroprotective effect, or to provide cerebroprotective effect,

5. treat cytotoxic T cell and natural killer cell-mediated apoptosisassociated with autoimmune disease and transplant rejection,

6. to prevent cell death of cardiac cells including heart failure,cardiomyopathy, viral infection or bacterial infection of heart,myocardial ischemia, myocardial infarct, and myocardial ischemia,coronary artery by-pass graft,

7. prevent and/or treat mitochondrial drug toxicity e.g. as a result ofchemotherapy or HIV therapy,

8. prevent cell death during viral infection or bacterial infection,

9. prevent and/or treat inflammation or inflammatory diseases,inflammatory bowel disease, sepsis and septic shock,

10. to prevent cell death from follicule to ovocyte stages, from ovocyteto mature egg stages (Nutt et al. 2005; Bergeron et al., 1998); andsperm (for example, methods of freezing and transplanting ovariantissue, artificial fecondation), or to preserve fertility in women andmen after chemotherapy, or to preserve fertility in females and malesanimals,

11. to prevent and/or treat, macular degenerescence and glaucoma,

12. to prevent and/or treat acute hepatitis, chronic active hepatitis,hepatitis-B, and hepatitis-C,

13. to prevent hair loss, and the hair loss due-to male-patternbaldness, radiation, chemotherapy or emotional stress,

14. to treat or ameliorate skin damage (due to exposure to high level ofradiation, heat, burns, chemicals, sun, and autoimmune diseases),

15. to prevent cell death of bone marrow cells in myelodysplasticsymdromes, or to treat pancreatisis, or to treat respiratory syndrome,

16. to treat and/or prevent death of gastrointestinal lining epithelialcells,

17. to treat osteoarthitis, rheumatoid arthritis, psoriasis,glomerulonephritis, atheroscerosis, and graft versus host disease,

18. to treat retinal pericyte apoptosis, retinal neurons apoptosis,glaucoma, retinal damages, macular degeneration resulting from ischemia,diabetic retinopaty, or other trauma.

According to a particular embodiment, the disease is a neurodegenerativedisease.

Exemplary neurodegenerative diseases include, but are not limited toHuntington's Disease (HD), Alzheimer's Disease (AD), aging and stroke.

Additional neurodegenerative diseases include Parkinson's disease,Multiple Sclerosis, ALS, multi-system atrophy, progressive supranuclearpalsy, fronto-temporal dementia with Parkinsonism linked to chromosome17 and Pick's disease.

The peptides of the present invention may be provided per se or as partof a pharmaceutical composition, where it is mixed with suitablecarriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the caspase 6 inhibitorypeptides accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the active ingredients to allowfor the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (caspase-6 inhibitory peptides) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., Huntington'sDisease) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to brain orblood levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, CA (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods

Peptide Synthesis and Dissolvent:

ED11 (GRKKRRQRRRPPQSSEIVLDGTDN—SEQ ID NO: 6) and TAT-only(GRKKRRQRRRPPQ—SEQ ID NO: 7) were synthesized by China-Peptides Ltd.with a purification level at over 95%. For the purified caspase activityassays, peptides were dissolved in DMSO. For the cell culture involvedexperiments, peptides were initially dissolved in cell-culture grade H₂Oand further diluted by phosphate-buffered saline (PBS) to the desiredconcentrations.

Purified Caspase Inhibition Assays:

For determining ED11 caspase-6 inhibition ability, the Caspase-Glo® 6assay (Promega) was carried out according to the manufacturer'sinstructions. Briefly, 0.1 U/ml Purified caspase-6 (Enzo life-science)was incubated with 0-12 μM Z-VEID-aminoluciferin in the presence of 10μM ED11 or DMSO as vehicle control, and luminescent signal of releasedaminoluciferin (as RLU) was detected using Synergy HT multi-modemicro-plate reader (Bio-Tek). For Michaelis-Menten curve plotting,initial velocity was calculated using linear regression of the progresscurve linear segment, adjusted to RLU/min. For caspase-3 inhibitioncapability, caspase-3 inhibitor drug detection kit (Abcam) was performedaccording to the manufacturer's instructions.

Brain Lysate Preparation and Western Blotting:

The rodents were placed under 12 hour light/dark conditions and housedin individually ventilated cages with ad libitum access to food andwater. For brain lysate preparation, FVB/N BACHD mice and theirwild-type littermates were euthanized by de-capitation. Striata weredissected and placed in lysis buffer (200 mM HEPES, 150 mM NaCl, 1 mMNa₃Vo₄, 5 mM EDTA, 1% NP-40, 0.5% DOC, 50 mM NaF) with proteaseinhibitors (Roche) for 1 hour on ice. Tissue debris was removed bycentrifugation at 20,000×g for 15 min at 4° C. Protein concentration wasdetermined by the BCA method (Pierce). 50 μg of lysate proteins wereexposed to 100 U/ml purified active human caspase-6 for 20 minutes in37° C. in the presence of 50 μM ED11 or DMSO as vehicle control. Lysateswere subsequently loaded on 7.5% SDS-PAGE gels and transferred to 0.45μM nitro-cellulose membranes. The membranes were probed with mouseanti-Htt 2166 (1:2000, Millipore), rabbit anti-Htt 4-19 (1:1000, CHDI),and mouse anti β-actin (1:10000, Sigma). Secondary antibodies used wereIRDye 800CW Goat anti-Mouse IgG, IRDye 800CW Goat anti-rabbit IgG andIRDye 680RD Goat anti-Mouse IgG, respectively. Fluorescent signal wasread using Odyssey imaging system.

Cell Culture:

Human neuroblastoma cells, SH-SY5Y cells (ATCC) were grown on tissueculture plates (Greiner) in Dulbecco's Modified Eagle's Medium (DMEM),supplemented with 10% fetal calf serum (FCS), 1% L-glutamine and 1% SPNantibiotics (Biological Industries Israel). Inducible 145Q-MHttexpressing PC12 cells were obtained from CHDI (by Coriell institute).Cells were grown in suspension in 75 cm³ culture flasks (Corning) inDulbecco's Modified Eagle's Medium (DMEM), supplemented with 15%Horse-serum, 2.5% FCS, 0.1 mg/ml G418 (Gibco), 0.1 mg/ml Zeocin(Invitrogen), 1% L-glutamine and 1% SPN antibiotics (BiologicalIndustries Israel). Both cell types were incubated at 37° C. in ahumidified atmosphere with 5% CO₂, and passaged twice a week.

Cell Proliferation and Cell Cycle Analysis:

SH-SY5Y cells were pre-incubated with 25 μM ED11 for one hour. Then, 10μM 5-bromo-2-deoxyuridine (BrdU) was added for 2 hours. Medium wasdiscarded and cells were fixed with 70% ethanol. DNA denaturation wasconducted with 1.5 M HCl exposure for 30 minutes. FITC conjugatedanti-BrdU antibody was used to mark incorporated BrdU. DNA staining wasperformed by using propidium iodide (PI). 15,000 cells per sample wereread using FACS-Calibur flow cytometer, and cell-cycle analysis wasconducted after doublet discrimination.

Glutamate Toxicity

To induce exogenous glutamate toxicity, SH-SY5Y cell were incubated withthe indicated concentrations of L-Glutamate (Sigma) for 16 hours, in thepresence of 20 μM ED11 or vehicle control. For the In-cell caspase-6activity assay, green FLICA caspase-6 assay (ImmunoChemistryTechnologies) was conducted according to the manufacturer'sinstructions. Briefly, cells were lifted by trypsin and adjusted to1.5×10⁶ cells/ml. Cells were incubated with FAM-VEID-FMK for 1 hour in37° C., washed with PBS and 5,000 cells per sample were analyzed byflow-cytometry. For apoptosis detection, Annexin V-FITC apoptosisdetection assay (Abcam) was conducted according to the manufacturer'sinstructions. Briefly, 2×10⁵ cells were incubated with Annexin V-FITCand PI for 5 minutes in R.T, and 15,000 cells per sample were analyzedby flow-cytometry. For viability assessment, medium was depleted andwashed twice with PBS, and alamar-blue dye (AbD serotec, 1/10 in culturemedium) was added to the cells, as instructed by the manufacturer.Fluorescence was monitored at 530-560 nm excitation wavelength and 590nm emission wavelength, and viability was calculated as percentage ofuntreated control.

Mutant Huntington Induced Toxicity

To induce mHtt expression, Inducible 145Q-mHtt expressing PC12 cellswere incubated with 25 μM ponasterone a (PA, Invitrogen) for theindicated time periods, in the presence of 25 μM ED11 or vehicle ascontrol. Cell viability was measured by alamar-blue as previouslydescribed. LDH release was measured by the LDH Cytotoxicity DetectionKit (Clonetech), following manufacturer's instructions. Briefly, PC12cells were grown on 6-well plate at a density of 7×10⁵ cells/ml, andsamples were taken at the indicated time points. Subsequently, sampleswere centrifuged, transferred to a 96 well plate and were incubated withthe reaction mixture for 30 minutes in the dark. The plate was read at492 nm and 690 nm as reference in Synergy HT multi-mode micro-platereader, and calculations were done in reference to a none-inducedcontrol. For FLICA caspase-6 assay, cells were adjusted to1.5×10⁶cells/ml and were incubated with FAM-VEID-FMK for 1 hour at 37°C. as described. Hoechst stain (ICT) was used as nucleuscounterstaining. The images were recorded with a fluorescence microscope(OLYMPUS). Image analysis was made by the Image-J software (Abramoff etal., 2004).

Animal Studies

To evaluate ED11 ability to provide protection from mHtt toxicityin-vivo, HD mice model BACHD was used. ED11 treatment commenced at theage of 5 weeks by a subcutaneously implanted mini-pump, which infusescontinuously for 28 days, at a dose of 4 mg/kg/day.

At the age of 9 weeks, motor coordination and strength were assessedusing the accelerating Rotarod test. Mice were trained for 3 consecutivedays on the accelerating rod (0 to 21 RPM in 4 minutes), 3 trials perday with a 2 hours inter-trial rest. On the fourth day Mice were testedon the accelerating rod for 3 consecutive tests, and best score wastaken for analysis.

Statistical Analysis

Statistical analysis was performed using GraphPad prism version 6.0.Statistical significance for differences between two groups wasevaluated by unpaired student's t-test. When three groups wereaddressed, statistical evaluation was made by one-way ANOVA followed byTukey's multiple-comparison post hoc test. When addressing time-pointsdependent alterations, two-way ANOVA with Tukey's multiple-comparisonpost hoc test was used. Data are presented as mean±SEM, and the level ofP<0.05 was accepted as statistically significant.

Results

EDII inhibits caspase-6 activity in a purified caspase-6 activity assay:To test the peptide ability to compete on caspase-6 activity, purifiedcaspase-6 and Z-VEID-Aminoluciferin (Caspase-6 Glo™, Promega) wereincubated with ED11, and bioluminescence signal was detected usingSynergyHT microplate reader (FIGS. 1A-1D). The peptide ED11(GRKKRRQRRRPPQSSEIVLDGTDN—SEQ ID NO: 6) significantly reduced caspase-6activity and using Michaelis-Menten curve it shows classic competitiveinhibition as Vmax stays approximately the same and Km shows a 3.3 foldincrease (FIG. 1C). In contrast, ED11 does not inhibit caspase-3 in afluorescent caspsae-3 inhibition assay (FIG. 1D).

A further experiment was performed to analyze the specificity of ED11.ED11 and Z-VAD-FMK, a synthetic pan caspase inhibitor used as control,were tested in the purified caspase-3/caspase-6 assay. Here it isdemonstrated that while Z-VAD-FMK inhibits activity of caspase-3 andcaspase-6, ED11 inhibits activity of caspase-6 but not caspase-3 (FIGS.1E-1F).

Interestingly, when three of the amino acids in ED11 in positions 17, 20and 23, to Histidine (H), the modified peptide was ineffective anddidn't inhibit the caspase 6 activity.

ED11 does not influence cell viability, proliferation or cell cyclestatus in a basal state: In order to provide a preliminary safetyevaluation, the influence of ED11 was tested on cell viability,proliferation and cell cycle state. ED11 was shown to have no effect onany of these parameters at a concentration up to 50 μM after 48 hoursincubation (FIGS. 2A-2C).

Evaluation of ED11 caspase-6 inhibition and protection against glutamatetoxicity in a neuroblastoma cell line: The protection of ED11 (25 μM)against glutamate was tested in a neuroblastoma cell line SH-SY5Y. Itwas found that ED11 significantly inhibited endogenous caspase-6activity that was induced by 17 mM glutamate, and protected the cellsfrom apoptosis (FIGS. 3A-3D).

ED11 protects inducible mHtt expressing PC12 cell-line from mHtttoxicity: In order to evaluate ED11 ability to protect cells from mHtttoxicity, PC12 cells harboring an inducible mHtt expression vector(CHDI), were placed under chronic serum deprivation stress, and inducedto express mHtt by 25 μM Ponasterone A (PA) for 72 hours. Cell viabilitywas measured by Alamar blue assay, cell death by LDH assay (Clonetech),and caspase-6 activity by FLICA in-cell caspase activity. In allparameters tested, ED11 was shown to significantly avert the toxiceffect of mHtt expression on the cells (FIGS. 4A-4D).

ED11 prevents mutant Huntingtin direct cleavage by caspase-6: To testED11's ability to interfere directly with caspase-6 cleavage of mHtt,brain striatum extracts, taken from BACHD mice were incubated withcaspase-6 for 20 minutes at 37° C. The addition of ED11 significantlyinhibited human mHtt fragmentation as indicated with specific anti-Httantibodies as illustrated in FIGS. 5A-5B (4-19 and MAB 2166).

Following administration of ED11, the present inventors showed that thepeptide was effective at protect the BACHD mice from motor deteriorationas was tested in the accelerating Rotarod test (FIGS. 7A-7C).

Relevance of amino acids X1 and X4: In order to test whether the aminoacids at position X1 and X4 are important for peptide activity, threeadditional peptides were synthesized which had substitutions atpositions X1 and X4 and their ability to inhibit caspase 6 was analyzed.

As illustrated in FIG. 8, substitutions at either position X1 and X4were detrimental to the ability of the peptide to inhibit caspase 6.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A method of treating Alzheimer's disease in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of an isolated peptidecomprising a Huntingtin (Htt) amino acid sequence being no longer than15 amino acids, wherein said Htt amino acid sequence comprises thesequence as set forth in SEQ ID NO: 2, wherein the peptide is capable ofspecifically inhibiting the activity of caspase 6, thereby treating theAlzheimer's disease.
 2. The method of claim 1, wherein said Htt aminoacid sequence is attached to a cell penetrating agent.
 3. The method ofclaim 2, wherein said cell penetrating agent is a cell penetratingpeptide agent.
 4. The method of claim 3, wherein said cell penetratingpeptide agent is attached to said Htt amino acid sequence via a linkingamino acid sequence.
 5. The method of claim 4, wherein said linkingamino acid sequence comprises a disulfide bridge.
 6. The method of claim4, wherein said linking amino acid sequence is attached to the Nterminus of said Htt amino acid sequence.
 7. The method of claim 5,wherein said linking amino acid sequence comprises the sequence SSE. 8.The method of claim 4, wherein said isolated peptide which comprisessaid cell penetrating peptide agent and said Htt amino acid sequence isno longer than 25 amino acids.
 9. The method of claim 1, wherein the Httamino acid sequence inhibits the activity of caspase 6 to a greaterextent than it inhibits the activity of caspase 3.